Negative-sense RNA viruses including filoviruses, rhabdoviruses and paramyxoviruses are the cause of a multitude of serious diseases in humans worldwide. Assembly and budding are important late events in the replication cycles of these viruses. While much has been learned regarding these dynamic and multifaceted stages of replication, host pathways that modulate virus assembly/budding continue to be identified. Assembly/budding of filoviruses and virus-like particles (VLPs) is driven largely by the matrix protein, VP40. We and others have shown that Late budding domains (L-domains) within VP40 are important for efficient budding, as they mediate interactions with host proteins (e.g. Nedd4 ubiquitin ligase) to facilitate virus-cell separation. Notably, it is becoming clear that host innate immune responses result in the induction of a myriad of proteins that modulate virus replication, budding, and pathogenesis. The innate immune response represents the first critical line of defense against viral pathogens, and importantly, ebolavirus and marburgvirus target key regulators of this response including macrophages/monocytes and dendritic cells. A better understanding of host innate immune interactions and responses to filoviruses will be crucial for developing therapeutics and vaccines to treat and prevent infection. Toward this end, we demonstrated recently that expression of interferon stimulated gene ISG15 resulted in inhibition of VP40 ubiquitination and subsequent egress of VP40 VLPs. Although identification of this previously undescribed mechanism of antiviral activity for ISG15 against ebolavirus was an important first step, our understanding of how ISG15 and associated ISGylation factors inhibit budding of ebolavirus and possibly marburgvirus remains incomplete. The overarching goals of this proposal are to determine whether Ebola VLPs can induce expression of IFN-regulated Herc5 E3 ligase through a TLR4-dependent pathway, whether Herc5 E3 ligase contributes to ISGylation of target proteins (host or viral) resulting in inhibition of ebolavirus budding, and whether host ubiquitination and ISGylation pathways play a competing role in modulating budding of marburgvirus VLPs. We believe that these studies will have a major impact on our understanding of the interplay between filovirus proteins and components of the innate immune system leading to modulation of virus egress and likely disease progression and severity. In addition, our findings will also impact our understanding of how manipulation of host immune response mediators may have therapeutic benefit, and may help to optimize immunological responses to potential vaccines (e.g. ebolavirus or marburgvirus VLPs) and/or vaccine components (e.g. ebolavirus or marburgvirus GP and VP40). PUBLIC HEALTH RELEVANCE: The filoviruses are deadly human pathogens and potential agents of bioterrorism for which no vaccines, nor antivirals exist. The innate immune system represents a first critical line of defense against these viral pathogens. A better understanding of how the host innate immune system recognizes, responds, and defends against filovirus infection will be critical for developing effective therapeutics and vaccines.